Cloning And Expression Of BAFF Gene And The Effects Of Boron In Drinking Water On The Tissue Expression In African Ostrich

B cell activating factor(BAFF) is one member of the tumor necrosis factor(TNF) family, which is critical for keeping the function of B cell. BAFF can promote B lymphocyte proliferation and activation when it binds to its receptor BAFF-R. BAFF could stimulate B lymphocytes’ proliferation, differentiation and antibody secretion. It is significance for promoting and regulating the survival and development of B lymphocytes. The production of BAFF by innate immune cells and some lymphocytes plays a central role in immune responses.The African ostrich, Struthio camelus, is the biggest bird on the earth, and its adaptability is very strong. In recent years, African ostrich breeding has been developed rapidly in many countries, including the USA, Australia, China, New Zealand, Israel, Canada, and South Africa because of its attractive economic value. The African ostrich has assumed significant economic importance as a farmed bird. Its B cells are targets of Marek’s disease virus(MDV), Newcastle disease virus(NDV), avian influenza virus(AIV), and infectious bursal disease virus(IBDV), etc. Immune suppression or even death will be caused by these infections in 3 months, which caused huge economic losses for ostrich breeding.Boron is an essential trace element which is widely distributed in nature. Boron has many biological effects. It involves in many physiological activities for some animals. The results have shown that boron is very important for the growth and metabolic process of some animals in recent years. It also showed that the quantum vis boron has trophic action on animals and the high dosage of boron was bad for growth, metabolism and other physiological function, and even toxic for men or animals.Regarding these as the starting point, we use molecular cloning, prokaryotic expression, and real-time PCR methods to study Africa ostrich BAFF gene, including the gene sequences, evolutionary relationship, tissue distribution and biological functions. At the same time, different dose of supplemental boric acid in drinking water have been offered to indicate the effects of boron on the BAFF gene tissue expression in African Ostrich. This study was designed to provide theory basis for the nutrition and immune toxicity of boron, molecular biology and immune defense mechanism of Africa ostrich. The main work and research results are as follows:1. Gene cloning and sequence analysis of Africa ostrich BAFFPrimers were designed by consulting other BAFF gene sequences published in Gen Bank. Total RNA was extracted from bursa of Fabricius of Africa ostrich as template and the BAFF gene of Africa ostrich was obtained by RT-PCR. After sequencing, homology analysis and phylogeny tree analysis of BAFF were conducted by MEGA 5.05 program and Signal P 4.1 server software. The results showed that the nucleotide sequence and the encoded protein were 867 bp and 288 amino acids in length, which contains a predicted transmembrane domain(TMD) of 23 amino acids and a putative furin protease cleavage site. As determined by BLAST, Os BAFF shows 52%, 51%, 50%, 50%, and 49% amino acid sequence identities with BAFFs from chicken(ch BAFF), quail(qs BAFF), duck(d BAFF), goose(g BAFF) and dove(do BAFF), respectively. Phylogenetic analysis showed that the phylogenetic tree was divided into three different branches with one containing all bird, one containing all mammalian, and the other with all fish proteins, and Os BAFF was clustered with bird being separated from other vertebrates. These results can provide references for the study of BAFF from the viewpoints of gene evolution and molecular biology in bird.2. Prokaryotic expression of BAFF gene in Africa ostrichBased on the complete Os BAFF sequence mentioned above, two primers Os-F and Os-R contained Eco R I and Hind III restriction sites, were designed to amplify the soluble part of Os BAFF. After digested with Eco R I and Hind III, the PCR product was cloned into the p ET-28 a vector, forming a sequence encoding a fusion protein composed of Os BAFF. The p ET-28a-Os BAFF plasmid was transformed into E. coli Rosset(DE3) expression strain, and soluble Os BAFF was optimally expressed and purified by Ni-sepharose. SDS-PAGE electro-phoresis and Western blotting showed a single band of recombinant Os BAFF which appeared with a molecular weight of approximately 32.2 kD.3. Tissue expression of Africa ostrich BAFF geneThe m RNA expression of Africa ostrich BAFF gene in 18 kinds of tissues including muscle, esophagus, stomach, small intestine, large intestine, pancreas, trachea, lung, testis, ovary, bone marrow, bursa of Fabricius, spleen, thymus, liver, kidney, heart and brain was analyzed by real-time PCR. The results showed that Os BAFF m RNA was widely expressed in most of the tested tissues except heart and brain, and its expression profiles varied among different tissues. High levels of BAFF expression were detected in bursa of Fabricius, thymus, spleen, and bone marrow; moderate levels in liver, lung, kidney, trachea, stomach, and intestine; and low levels in the other tested tissues. Different expression levels of Africa ostrich BAFF gene could indicate that its main functions in these tissues were different.4. Biological activity of Africa ostrich Os BAFFMammalian BAFF and other birds’ BAFF are a survival factor for B cells. To test whether this also holds true for African ostrich system, bursal lymphocyte cells of African ostrich were isolated and treated with Os BAFF. Freshly isolated African ostrich bursal lymphocytes were cultured with purified Os BAFF or Os BAFF + 2 μg/ml PMA for 24 h.WST-8 assay revealed that the numbers of living cells were higher in Os BAFF treated cultures compared to the control. A dose-dependent response to Os BAFF treatment was observed. A dose-dependent response to Os BAFF or Os BAFF + 2 μg/ml PMA treatment was clearly observed, and saturation was reached the highest if used at 12 μg/ml Os BAFF or 16 μg/ml Os BAFF + 2 μg/ml PMA. The negative control PBS and BSA had no survival effect on bursal B cells. The results of mouse B cells stimulation showed that Os BAFF could stimulate the survival/proliferation of mouse B cells in vitro and its effects under different doses were similar with the results of African ostrich bursal lymphocytes. The results of LPS stimulation showed that the expression levels of Os BAFF in African ostrich bursal lymphocyte cells after 6, 12, 24, and 48 h were higher in the LPS stimulated group. The m RNA expression increased from 6h and up-regulated sharply to a maximum at 24 h, and remained at a high level until 48 h(p < 0.01) compared with the control group.5. The effects of boron in drinking water on the tissue expression of BAFF gene in African ostrichIn order to study the effects of boron in drinking water on the tissue expression of BAFF gene in African ostrich. Forty-eight 1 day old African ostrich chicks were divied into 6 groups randomly. In each group, drinking water was in different concentration of boric acid, 0 mg/L(contral group), 40 mg/L, 80 mg/L, 160 mg/L), 320 mg/L and 640 mg/L, respectively, which were supplied uninterrupted for 90 days. The tissues such as bone marrow, thymus, bursa of Fabricius, spleen, lung, kindey, liver and lung were collected at 1 day, 45 days and 90 days after added boric acid in drinking water. The m RNA expression of Africa ostrich BAFF gene in tissues was analyzed by real-time PCR. The results showed that the tissue expression of BAFFgene in Africa ostrich is dose dependent with boron in drinking water. When the dose of boron in drinking water is low, the expression of BAFF gene in the eight detected tissues increased along with the increased dose of boron added in drinking water. And when the dose of boron added in drinking water increased to a certain extent, the expression of BAFF gene in the tissues decreased along with the increased dose of boron added in drinking water. For 45 days old Africa ostrich, the highest level of BAFF gene expression was the 160 mg/L boron added in drinking water experimental group in bone marrow, thymus, spleen and lung, the highest level of BAFF gene expression was the 80 mg/L boron added in drinking water experimental group in bursa of Fabricius, liver and kindey, and they were all significantly higher than that of control group(P < 0.05). The highest level of BAFF gene expression was the 80 mg/L boron added in drinking water in stomach, and there was no significant difference compared with control group. For 90 days old Africa ostrich, the highest level of BAFF gene expression was the 80 mg/L boron added in drinking water experimental group in bone marrow, bursa of Fabricius, liver, spleen, thymus and kindey,the highest level of BAFF gene expression was the 160 mg/L boron added in drinking water experimental group in lung, and they were all significantly higher than that of control group(P < 0.05). The highest level of BAFF gene expression was the 160 mg/L boron added in drinking water in stomach, and there was no significant difference compared with control group.